Nitroxides as Protectors against Oxidative Stress Summary NNitroxides are proving to have broad utility in a number of disease processes and/or conditions that represent excessive oxidative stress. The fact that nitroxides exert activity over such a range of conditions speaks to the importance of free radical reactions in tissue. Likewise, it is becoming apparent that free radicals are important in normal molecular signaling pathways and related gene expression. In collaborative studies, the effects of chronic administration of Tempol (supplemented in food) of two mouse models that exhibit neurodegeneration and/or neurological damage have been evaluated. Tempol was shown to be highly protective in an experimental autoimmune encephalomyelitis (EAE) mouse model. The EAE mouse model is an acute or chronic demyelinating autoimmune disease whose clinical manifestations of paralysis and quadriparesis that closely resemble those observed in Multiple Sclerosis (MS) patients. Pre-treatment of mice with Tempol markedly decreased paralysis. Likewise, Tempol treatment at the onset of disease decreased paralysis as well indicating that Tempol might have utility in both treating and preventing MS. Studies are ongoing exploring the mechanism of the protection. We continue to search for the mechanism(s) of how long-term administration of Tempol (in the food or drinking water) results in dramatic weight reduction and a decrease in spontaneous tumor incidence in mice. Gene expression studies were conducted by evaluating tissue taken from age-matched control mice and mice on Tempol food supplementation for 1 month or 1 year have allowed us to focus on several gene families that might be important in the effect. Glutathione metabolism genes were up-regulated as well as genes associated with fat synthesis and storage. Genes coding for proteins in the Nrf2 system (with provide protection against free radicals) were also up-regulated. Currently, we are determining if protein levels in tissue taken from Tempol-treated mice are elevated. We have also evaluated metabolic products in the urine of mice consuming Tempol and have found a host of metabolites. Data evaluation is currently ongoing. Lastly, in a related project, we evaluated a different antioxidant (resveratrol) and its metabolic product piceatannol as protectors against oxidative stress (hydrogen peroxide, radiation). Piceatannol was found to provide concentration-dependent protection against hydrogen peroxide induced cytotoxcity;whereas, resveratrol markedly enhanced the cytotoxicitiy. Neither agent afforded protection against ionizing radiation in vitro or in vivo, suggesting that these agents should not be evaluated clinically.